A kind of Monascus purple ester synthase lip05, coding gene and application thereof
A technology of LIP05, encoding gene, applied in the field of genetic engineering, can solve problems such as no reports yet
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Embodiment 1
[0043] Cloning of the gene encoding ester synthase LIP05
[0044] 1 Culture of Monascus purple
[0045] Under aseptic conditions, Monascus purple was inoculated into a 300mL Erlenmeyer flask containing 100mL of fermentation medium, and cultured on a shaker at 30±1°C and 150±10r / min for 4-8d. The fermentation medium is composed of glucose 70g / L, soybean cake powder 10g / L, MgSO 4 0.20g / L, NaNO 3 2.0g / L, NaH 2 PO 4 1.0g / L, adjust the pH to 4.5, and sterilize at 115°C for 20min.
[0046] 2 Extraction of Monascus purple total RNA
[0047] The above-mentioned Monascus violaceum cultured for 6 days was sampled, and the total RNA was extracted using the BIOMIGA Fungal RNA Extraction Kit. Specific steps are as follows:
[0048] (1) Weigh 100mg of fungal culture into a 1.5mL or 2.0mL centrifuge tube, freeze in liquid nitrogen, grind the fungus into powder with a grinding pestle (if possible, put the tissue in a mortar, and grind it into powder with liquid nitrogen , quickly t...
Embodiment 2
[0085] Construction of Escherichia coli engineering strain expressing ester synthase LIP05
[0086] Linearization of 1pCold-TF vector
[0087] Primers were designed to linearize the pCold-TF vector by PCR. The primers were designed as follows: Forward primer: 5'-ctgcagtctagataggtaatc-3' (SEQ ID No.5)
[0088] Reverse primer: 5'-accaccactacctcgtggca-3' (SEQ ID No.6)
[0089] The PCR reaction system is shown in Table 4.
[0090] Table 4 PCR reaction system of linearized vector pCold-TF
[0091] Reagent Volume (μl) wxya 2 o
21.0 dNTP Mixture (2.5mM each) 3.0 10×Ex Taq Buffer 3.0 Forward primer (10μM) 0.6 Reverse primer (10μM) 0.6 pCold-TF vector 0.8 Q5 DNA Polymerase (5U / μl) 1.0 total 30.0
[0092] The PCR amplification reaction procedure is as follows:
[0093]
[0094] The amplified DNA band is used for plasmid construction after DNA purification.
[0095] 2 Plasmid construction
[0096] pass II car...
Embodiment 3
[0103]Synthesis of Ethyl Hexanoate and Ethyl Octanoate Catalyzed by Ester Synthase LIP05 in Liquid Aqueous Phase System
[0104] Induced expression of 1 ester synthase LIP05
[0105] Pick the verified correct transformants, transfer them to LB liquid test tubes containing appropriate ampicillin antibiotics, culture overnight at 37±1°C, then inoculate a 300mL Erlenmeyer flask containing 100mL LB medium with an inoculum of 1% (v / v), shake The bed was cultured at 37±1° C. and 200 rpm for 3 hours, then the inducer IPTG with a final concentration of 0.5 mM was added, and cultured at 20±1° C. and 200±10 rpm for 20 hours.
[0106] 2 Preparation of crude enzyme solution of ester synthase LIP05
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